Two siblings presenting with novel ADA2 variants, lymphoproliferation, persistence of large granular lymphocytes, and T-cell perturbations.

The presence of large granular lymphocytes has been reported in patients with ADA2 deficiency and T-LGL leukemia. Here we describe two siblings with novel ADA2 variants, expanding the mutational spectrum of ADA2 deficiency. We show that lymphoproliferation, persistence of large granular lymphocytes, T-cell perturbations, and activation of PI3K pathway, measured by means of phosphorylation levels of S6, are detectable in DADA2 patients without T-LGL leukemia.

Comparison of multiple definitions for ventilator-associated pneumonia in patients requiring mechanical ventilation for non-pulmonary conditions: preliminary data from PULMIVAP, an Italian multi-centre cohort study.

OBJECTIVES: To compare intensivist-diagnosed ventilator-associated pneumonia (iVAP) with four established definitions, assessing their agreement in detecting new episodes. METHODS: A multi-centric prospective study on pulmonary microbiota was carried out in patients requiring mechanical ventilation (MV). Data collected were used to compare hypothetical VAP onset according to iVAP with the study consensus criteria, the European Centre for Disease Control and Prevention definition, and two versions of the latter adjusted for leukocyte count and fever. RESULTS: In our cohort of 186 adult patients, iVAPs were 36.6% (68/186, 95% confidence interval 30.0-44.0%), with an incidence rate of 4.64/100 patient-MV-days, and median MV-day at diagnosis of 6. Forty-seven percent of patients (87/186) were identified as VAP by at least one criterion, with a median MV-day at diagnosis of 5. Agreement between intensivist judgement (iVAP/no-iVAP) and the criteria was highest for the study consensus criteria (50/87, 57.4%), but still one-third of iVAP were not identified and 9% of patients were identified as VAP contrary to intensivist diagnosis. VAP proportion differed between criteria (25.2-30.1%). CONCLUSIONS: Caution is needed when evaluating studies describing VAP incidence. Pre-agreed criteria and definitions that capture VAP's evolving nature provide greater consistency, but new clinically driven definitions are needed to align surveillance and diagnostic criteria with clinical practice.

Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2-rearranged BCP-ALL.

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Drug-induced immunophenotypic modulation in childhood ALL: implications for minimal residual disease detection.

Assessment of minimal residual disease (MRD) by flow cytometry is considered to be based on the reproducibility of the leukemic immunophenotype detected at diagnosis. However, we previously noticed modulation of surface antigen expression in acute lymphoblastic leukemia (ALL) during the early treatment. Hence, we investigated this in 30 children with B-cell precursor ALL consecutively enrolled in the AIEOP-BFM ALL 2000 protocol. Quantitative expression of seven antigens useful in MRD monitoring was studied at diagnosis and compared to that measured at different time points of remission induction therapy. Downmodulation in the expression of CD10 and CD34 occurred at follow-up. By contrast, upmodulation of CD19, CD20, CD45RA, and CD11a was observed, while the expression of CD58 remained stable. Despite this, we could unambiguously discriminate leukemic cells from normal residual B cells. This holds true when bone marrow (BM) samples from similarly treated T-ALL patients, but not from healthy donors, were used as reference. Our results indicate that immunophenotypic modulation occurs in ALL during the early phases of BFM-type protocols. However, the accuracy of MRD detection by flow cytometry seems not negatively affected if adequate analysis protocols are employed. Investigators should take this phenomenon into account in order to avoid pitfalls in flow cytometric MRD studies.

CD40 ligand-stimulated B cell precursor leukemic cells elicit interferon-gamma production by autologous bone marrow T cells in childhood acute lymphoblastic leukemia.

Childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, collected from bone marrow (BM) at diagnosis, were cultured, after thawing, on allogeneic human bone marrow stroma (HBMS) for 48 h in the presence of a soluble trimeric CD40 ligand (stCD40L) molecule. HBMS maintained leukemic cells viability in all tested cases (mean viability 85%). Under these culture conditions we noticed upregulation or de novo expression of costimulatory molecules CD40, CD80 (B7-1) and CD86 (B7-2) in 22/22, 15/23 and 21/23 cases, respectively. Upregulation, in terms of fluorescence intensity, was also observed in the expression of MHC I, MHC II, CD54 (ICAM 1) and CD58 (LFA 3) molecules. HBMS alone, although to a lesser extent, was able to induce modulation of these molecules, but not CD80, in a similar proportion of cases. Neither stCD40L nor HBMS induced modulation of CD10 and CD34 molecules. Moreover, in 4/4 tested cases, stCD40L-stimulated ALL cells were able to induce allogeneic T cells proliferation. To evaluate whether leukemia-reactive T cells were detectable in the BM of ALL patients at diagnosis, stCD40L-stimulated ALL cells were co-cultured with autologous T cells (ratio 1:1), isolated from BM at diagnosis, for 4 days and a 24 h ELISPOT assay was applied to detect the presence of interferon-gamma (IFN-gamma)-producing cells. In four of seven cases IFN-gamma-producing cells were detected with frequencies of 1/900, 1/1560, 1/2150 and 1/1575 autologous T cells. These data confirm that stCD40L exposure can activate the antigen-presenting cell (APC) capacity of BCP-ALL cells cultured on HBMS and that ELISPOT assay can be used to measure the frequency of leukemia-reactive autologous T cells in the BM of ALL patients even after short-term culture with stCD40L-stimulated ALL cells.

CD99 expression in T-lineage ALL: implications for flow cytometric detection of minimal residual disease.

Expression of CD99 is higher on immature than on mature T cells. We postulated that this marker could be used to assess minimal residual disease (MRD) in T-lineage acute lymphoblastic leukemia (T-ALL). In diagnostic bone marrow (BM) samples from 27 children with T-ALL, expression of CD99 on leukemic lymphoblasts by flow cytometry was in median 7.7 times higher than on normal T lymphocytes from within the same sample. In 85% of cases, leukemic MFI values were higher than the mean MFI+2 s.d. of normal populations. We applied CD99 to study MRD in 39 follow-up samples from 15 consecutive T-ALL patients, and compared the results with those obtained with the well-established MRD-marker terminal deoxynucleotidyl transferase (TdT). Either antibody was combined in four-color flow cytometry with CD7, surfaceCD3, and cytoplasmicCD3. We found that CD99 was a valid complement to TdT in quantifying T-ALL MRD. Given a considerable interpatient variability, CD99 could be favorably used in nine patients, and TdT in other five patients. Both approaches showed a similar very low nonspecific background throughout 12 weeks from diagnosis (in median 0.002% of nucleated BM cells in patients with non-T ALL). We conclude that CD99 is a highly informative tool for MRD detection in T-ALL, bearing the advantage of surface expression in contrast to TdT.

Purified autologous grafting in childhood acute lymphoblastic leukemia in second remission: evidence for long-term clinical and molecular remissions.

Autologous transplantation is a treatment option for relapsed childhood acute lymphoblastic leukemia (ALL) in second complete remission (CR2) when a suitable donor is not available. In an attempt to prevent relapses originating from graft leukemic contamination, the experimental protocol of in vitro purification of leukapheretic products with monoclonal antibodies (MoAbs), previously reported for adults, was adopted in 11 of 12 consecutive patients (median age, 9 years) with B cell precursor ALL in CR2 after late relapse (median, 37; range, 31-51 months after the onset) enrolled between July 1997 and July 1999 at a single pediatric center. At a median of 12 days after the mobilizing chemotherapy followed by G-CSF, a median of 13.9 (range, 5.9-18.7) x 10(6) CD34+ cells/kg were collected from each patient and a median of 7.5 (range, 4.1-12.6) x 10(6) CD34+ cells/kg underwent the purification procedure. The first step of immunorosetting allowed a one-log reduction of the total cell count, by eliminating more than 90% of the CD11b+ cells; the second step, performed after incubation with anti-CD19 MoAbs, allowed the depletion of 99% (range, 93-100) of the CD19+ cells, kept within the magnetic field of the immunodepletion column, with a median recovery of 73% (range, 55-87) of the collected CD34+ cells. Molecular analysis assessed the in vitro eradication of detectable leukemic cells. A median reinfusion of 5.2 (range, 3.2-9.1) x 10(6) CD34+ cells/kg for each patient (median viability, 90%), after conditioning with the 'TBI-VP16-CY' regimen, allowed prompt engraftment and immunological reconstitution; no patients experienced severe transplant-related toxicity or major infections. One patient relapsed 7 months after transplantation, while 10 patients are alive in clinical and molecular remission, at a median follow-up of 29 months (range, 15-40) (2-year EFS, 89%, s.e. 9). In conclusion, the procedure proved to be reproducible for pediatric purified autografting, highly efficient concerning stem cell recovery and depletion of leukemia-lineage specific cells, and promising in terms of final outcome.

BIOMED-I concerted action report: flow cytometric immunophenotyping of precursor B-ALL with standardized triple-stainings. BIOMED-1 Concerted Action Investigation of Minimal Residual Disease in Acute Leukemia: International Standardization and Clinical Evaluation.

The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%). Parallel flow cytometric studies in different laboratories finally resulted in highly concordant results (>90%) for all five antibody combinations, indicating the high reproducibility of our approach. In conclusion, the technique presented here with triple-labelings forms an excellent basis for standardized flow cytometric MRD studies in multicenter international treatment protocols for precursor-B-ALL patients.

BIOMED-1 concerted action report: flow cytometric characterization of CD7+ cell subsets in normal bone marrow as a basis for the diagnosis and follow-up of T cell acute lymphoblastic leukemia (T-ALL).

The European BIOMED-1 Concerted Action was initiated in 1994 to improve and standardize the flow cytometric detection of minimal residual disease (MRD) in acute leukemia (AL). Three different protocols were defined to identify the normal subsets of B, T and myeloid cells in bone marrow (BM), and were applied to the different types of AL in order to study aberrant immunophenotypes. Using sensitive acquisition methods ('live gate') T cell subsets in normal BM could be identified with five triple-stains: CD7/CD5/CD3, CD7/CD4/CD8, CD7/CD2/CD3, CD7/CD38/CD34 and TdT/CD7/surface or cytoplasmic (cy)CD3 (antibodies conjugated with FITC/PE/PECy5 or PerCP, respectively). The identification of T cell subsets in BM allowed definition of 'empty spaces' (ie areas of flow cytometric plots where normally no cells are found). All studied T-ALL cases (n = 65) were located in 'empty spaces' and could be discriminated from normal T cells. The most informative triple staining was TdT/CD7/cyCD3, which was aberrant in 91% of T-ALL cases. In most cases, two or more aberrant patterns were found. Apparently the immunophenotypes of T-ALL differ significantly from normal BM T cells. This is mostly caused by their thymocytic origin, but also the neoplastic transformation might have affected antigen expression patterns. Application of the five proposed marker combinations in T-ALL contributes to standardized detection of MRD, since cells persistent or reappearing in the 'empty spaces' can be easily identified in follow-up BM samples during and after treatment.

Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal residual disease in precursor-B-ALL.

During the last two decades, major progress has been made in the technology of flow cytometry and in the availability of a large series of monoclonal antibodies against surface membrane and intracellular antigens. Flow cytometric immunophenotyping has become a diagnostic tool for the analysis of normal and malignant leukocytes and it has proven to be a reliable approach for the investigation of minimal residual disease (MRD) in leukemia patients during and after treatment. In order to standardize the flow cytometric detection of MRD in acute leukemia, a BIOMED-1 Concerted Action was initiated with the participation of six laboratories in five different European countries. This European co-operative study included the immunophenotypic characterization and enumeration of different precursor and mature B cell subpopulations in normal bone marrow (BM). The phenotypic profiles in normal B cell differentiation may form a frame of reference for the identification of aberrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precursor-B-ALL) and may therefore be helpful in MRD detection. Thirty-eight normal BM samples were analyzed with five different pre-selected monoclonal antibody combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19, CD19/CD34/CD45 and TdT/CD10/CD19. Two CD19- immature subpopulations which coexpressed B cell-associated antigens were identified: CD34+/CD22+/CD19- and TdT+/CD10+/CD19-, which represented 0.11 +/- 0.09% and 0.04 +/- 0.05% of the total BM nucleated cells, respectively. These immunophenotypes may correspond to the earliest stages of B cell differentiation. In addition to these minor subpopulations, three major CD19+ B cell subpopulations were identified, representing three consecutive maturation stages; CD19dim/CD34+/TdT+/CD10bright/CD22dim/CD45dim /CD38bright/CD20- (subpopulation 1), CD19+/CD34-/TdT-/CD10+/CD22dim/CD45+/CD38bright/ CD20dim (subpopulation 2) and CD19+/CD34-/TdT-/CD10-/CD22bright/CD45bright/ CD38dim/CD20bright (subpopulation 3). The relative sizes of subpopulations 1 and 2 were found to be age related: at the age of 15 years, the phenotypic precursor-B cell profile in BM changed from the childhood 'immature' profile (large subpopulations 1 and 2/small subpopulation 3) to the adult 'mature' profile (small subpopulation 1 and 2/large subpopulation 3). When the immunophenotypically defined precursor-B cell subpopulations from normal BM samples are projected in fluorescence dot-plots, templates for the normal B cell differentiation pathways can be defined and so-called 'empty spaces' where no cell populations are located become evident. This allows discrimination between normal and malignant precursor-B cells and can therefore be used for MRD detection.

Peripheral blood cells from children with RASopathies show enhanced spontaneous colonies growth in vitro and hyperactive RAS signaling.

Germline mutations in genes coding for molecules involved in the RAS/RAF/MEK/ERK pathway are the hallmarks of a newly classified family of autosomal dominant syndromes termed RASopathies. Myeloproliferative disorders (MPDs), in particular, juvenile myelomonocytic leukemia, can lead to potentially severe complications in children with Noonan syndrome (NS). We studied 27 children with NS or other RASopathies and 35 age-matched children as control subjects. Peripheral blood (PB) cells from these patients were studied for in vitro colony-forming units (CFUs) activity, as well as for intracellular phosphosignaling. Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects. We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies. Our findings demonstrate for the first time that PB cells isolated from children suffering from NS or other RASopathies without MPD display enhanced BFU-E and CFU-GM colony formation in vitro. The biological significance of these findings clearly awaits further studies. Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients.

Genetic modification of human T cells with CD20: a strategy to purify and lyse transduced cells with anti-CD20 antibodies.

A retroviral vector has been constructed that contains the human CD20 cDNA under the control of the Moloney murine leukemia virus (Mo-MuLV) LTR. Freshly isolated mononuclear cells are infected for three consecutive days in the presence of PHA and hrlL-2, and a mean 15.9% of the cells (range, 6.5 to 31.7%) acquire a CD3+CD20+ phenotype. Transduced T lymphocytes grow and expand in vitro for up to 3 weeks like mock-infected cells and, as observed for the T lymphoblastoid CEM cell line, CD20 expression is maintained for several months with no change in the growth curve of the cells. CD20-expressing CEM and fresh T lymphocytes can be positively immunoselected on columns using different anti-CD20 antibodies. Exposure to monoclonal chimeric anti-CD20 IgG1(kappa) Rituximab antibody (Roche), in the presence of complement, results in effective and rapid killing of the transduced CD3+CD20+ human T cells in vitro. This approach represents a new and alternative method to gene manipulation with "suicide" genes for the production of drug-responsive T cell populations, a crucial step for the future management of graft-versus-host disease in bone marrow transplant patients.

Peripheral blood stem cell collection in children with acute leukemia: effectiveness of the 'DIAVE' mobilizing regimen.

Few experiences of peripheral blood (PB) hematopoietic stem cell mobilization for autologous transplantation have been reported to date in children with acute leukemia (AL). The five-drug-chemotherapy 'DIAVE' (dexamethazone, idarubicine, cytosine-arabinoside, vincristine, etoposide), followed by G-CSF, previously reported as consolidation, was adopted as a mobilization regimen in 29 children (median age: 8 years, range: 3-21; median weight: 34 kg, range: 15-73) with ALL in second remission (CR2: 21), in CR3 (2) or ANLL in CR1 (6). A median peak of 94 x 10(6) CD34(+)cells/l (range: 10-604) was reached at a median time of 12 days (range: 10-18) after the beginning of the mobilizing regimen, which was well tolerated. A median of 10.9 x 10(6) CD34(+)cells/kg (range: 2.4-56.6) were collected in 25 patients (86%), approaching 40 x 10(6)/l CD34(+) cells in the PB (ALL in CR2: 20/21, in CR3: 0/2; ANLL: 5/6) by means of one (20) or two (5) leukaphereses; a median of 2.5 blood volumes was processed. Patients with ANLL mobilized more cells than patients with ALL; moreover, the shorter the interval between remission and mobilizing therapy, the higher was the yield. The products collected underwent purification, aiming at achieving complete removal of possibly contaminating leukemic cells, in 21 cases; also, unmanipulated aliquots were stored as rescues for all but one patient. All the 23 patients undergoing transplantation engrafted (ANC >0.5 x 10(9)/l) at a median of 12 days. In conclusion, the DIAVE regimen compares favorably with conventional mobilizing regimens, usually containing cyclophosphamide, in terms of low toxicity, collection time predictability, and efficacy, as shown by the high proportion of patients mobilizing, the large amounts of stem cell collected by means of one or two leukaphereses only, and the prompt engraftment after infusion.

Allogeneic bone marrow stem cell transplantation following CD34+ immunomagnetic enrichment in patients with inherited metabolic storage diseases.

T-cell depletion is an essential step in reducing the risk of graft-versus-host disease (GVHD) in patients with inherited metabolic storage diseases (IMSD) undergoing hematopoietic stem cell transplantation. This goal can be achieved either by selective removal of T cells or by positive selection of CD34+ cells. Large-scale preparations of purified CD34+ cells from bone marrow products have not been extensively described. We report our results with bone marrow CD34+ cell enrichment using the CliniMACS system in eight children with IMSD. The median recovery of positively selected CD34+ cells was 46.2% with a purity of 97.5%, and a residual T cell content of 0.04 x 10(6). A median of 5.5 x 10(6)/kg of CD34+ cells was infused. All patients engrafted at a median time of 12 days and none of the patients developed GVHD. This method is technically feasible and can be successfully used to transplant children with IMSD.

Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia.

To diagnose juvenile myelomonocytic leukemia (JMML) is sometimes challenging, because around 10% of patients lack molecular abnormalities affecting Ras-MAPK (mitogen-activated protein kinase) pathway and other diseases such as cytomegalovirus infection can mimic clinical signs of JMML. In order to validate a phospho-specific flow cytometry assay assessing phospho-signal transducer and activator of transcription factor 5 (p-STAT5) as a new diagnostic tool for JMML, we examined 22 samples from children with JMML and 47 controls. CD33+/CD34+ cells from 22 patients with JMML showed hyperphosphorylation of STAT5 induced by sub-saturating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF). Using a training set of samples (11 JMML and 23 controls), we identified a threshold for p-STAT5-positive after stimulation with 0.1 ng/ml GM-CSF (17.17%) that discriminates JMML from controls. This threshold was validated in an independent series (11 JMML, 24 controls and 7 cases with diseases other than JMML) where we demonstrated that patients with JMML could be distinguished from other subjects with a sensitivity of 91% (confidence interval (CI) 59-100%) and a specificity of 87% (CI 70-96%). Positive and negative predictive values were 71% (CI 42-92%) and 96% (CI 82-100%), respectively. In conclusion, flow cytometric p-STAT5 profiling is a reliable diagnostic tool for identifying patients with JMML and can contribute to consistency of current diagnostic criteria.

Autologous purified peripheral blood SCT in childhood low-risk relapsed ALL.

The treatment of childhood B-cell-precursor ALL after isolated-extramedullary or late relapse is controversial. Most approaches are based on chemotherapy or allogeneic transplantation. The aim of this report is to assess the long-term outcome of children with 'low-risk' relapsed ALL treated according to a prospective purified auto-transplantation protocol. From January 1997 to March 2004, at a single pediatric Center, 30 ALL consecutive children, lacking an HLA-identical sibling, were treated according to the autologous purified peripheral blood stem cell protocol after isolated-extramedullary (7) or late medullary (24) relapse. After the 'DIAVE' mobilizing regimen a median of 11.6 × 10(6)CD34+/Kg (range 3.9-27.4) were collected. Leukaphereses were depleted by 99% of CD19+cells (range 98-100) by means of a double step immunological purification. The conditioning regimen included TBI. No early severe complications nor transplant-related deaths occurred; late effects, as expected, mostly consisted in endocrinological issues and were assessed at a median follow-up of 8.5 years. Five-year-EFS and survival were 68.5% (s.e. 7.9) and 85.7% (s.e. 5.9), respectively, for the 35 eligible patients and 70.0% (s.e. 8.4) and 86.7% (s.e. 6.2) for the 30 patients actually transplanted as per protocol. The outcome of this series favorably compares with historical data regarding both autologous transplantation and standard salvage chemotherapy.